Proteoglycans are composed of glycosaminoglycan (GAG) side chains covalently attached to a protein core and are components of the extracellular matrix. These proposed experiments center on the thesis that proteoglycans are produced and secreted by granulosa cells in response to hormones and contribute to cellular differentiation. Those proteoglycans are associated with granulosa cell surfaces in specific binding sites and influence the binding of gonadotropins during folliculogensis. They also may modulate events associated with cumulus expansion. Composition of the proteoglycans and GAGs varies with follicle size and may vary with follicle health, and differences may exist in the chemical nature of granulosa-associated GAGs compared to GAGs soluble in follicular fluid. The following Specific Aims are proposed: (1) To chemically identify the disaccharide composition of GAGs in bovine follicular fluid (FF) and from granulosa membranes from the same FF samples in relation to follicle size and estrogen concentrations, (2) To isolate, purify and radiolabel chondroitin sulfate and heparan sulfate from bovine follicular fluid in mass quantities so sufficient quantities are available for binding assays and cultures of rat granulosa and bovine cumuli, (3) To chemically desulfate chondroitin and heparan from Specific Aim #2, followed by radiolabeling. The resulting hybrids will be used to evaluate whether the degree of sulfation affects the binding to granulosa as well as cultures of granulosa (Aim #5) or cumuli (Aim #6), (4) To characterize the binding site(s) for GAGs in solubilized granulosa membranes using affinity chromatography, electrophoresis, and testing their enzyme susceptibilities, (5) To evaluate the effects of follicular GAGs (Aim #2) and desulfated GAGs (Aim #3) on steroid production, proteoglycan/GAG biosynthesis, GAG binding and gonadotropin binding in cultured rat granulosa cells, (6) To determine whether follicle GAGs (Aim #2) and desulfated GAGs (Aim #3) affect GAG binding, hyaluronic acid production and cumulus expansion in response to FSH. These experiments will relate follicle size and follicle health to composition of follicular fluid GAGs and GAGs in granulosa membranes. In addition, structural features of GAGs to affect their binding will be examined and components on granulosa which may modulate GAG binding will be characterized. In vitro cultures of rat granulosa and bovine cumuli will enable experiments to be performed to assess hormonal regulation of proteoglycan/GAG production and determine how GAGs interacting with the cells modulate gonadotropin effects during folliculogenesis.